AU2019101814A4 - ESTABLISHMENT METHOD OF CELL LINE FOR PREPARING ANTI-EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) FULLY-HUMANIZED MONOCLONAL ANTIBODY (mAb) - Google Patents

Abstract

OF THE DISCLOSURE The present disclosure provides an establishment method of a cell line for preparing an anti epidermal growth factor receptor (EGFR) fully-humanized monoclonal antibody (mAb), where dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary (CHO) cell lines CHO/DHFR- are subjected to POOL screening after undergoing transfection, and the POOL screening includes: stage 1, using an HT-free SFM4CHO medium to screen out a cell line integrated with a heavy chain (HC) gene, and using Blasticidin in the medium to screen out a cell line integrated with a light chain (LC) gene; and stage 2, adding MTX to increase copy numbers of the LC gene and the HC gene that are integrated into a genome of a cell line, thereby increasing an expression level. A stable cell line with an expression level of 2.0 g/L to 3.0 g/L suitable for large-scale production processes is cloned and screened out, and the SEC, pI-cEF, deglycosylated molecular weight, and cell-based antibody activity of a product are improved.

Description

ESTABLISHMENT METHOD OF CELL LINE FOR PREPARING ANTI-EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) FULLY-HUMANIZED MONOCLONAL ANTIBODY (mAb)

TECHNICAL FIELD

[01] The present disclosure relates to the technical field of biomedicine, and in particular to an establishment method of a cell line for preparing an anti-epidermal growth factor receptor (EGFR) fully-humanized monoclonal antibody (mAb).

BACKGROUND ART

[02] mAb is a highly homogenous antibody produced by a single B cell clone, which specifically targets an antigenic epitope and has indications mainly of tumors, autoimmune diseases (AIDs), infectious diseases, and other aspects. Since the first therapeutic mAb Rituxin worldwide was approved for marketing by the Food and Drug Administration (FDA) in 1997, mAb has undergone rapid development in the global biomedical field in just 20 years and have become one of the research hotspots in recent years.

[03] In recent years, a number of companies and research institutions researching therapeutic mAb drugs have also emerged in China, where therapeutic antibodies targeting EGFR are particularly concerned. EGFR is a transmembrane glycoprotein, which is highly expressed in solid tumors. EGFR-positive tumors have the characteristics of high malignancy and strong invasiveness, and an expression level of EGFR is related to tumor prognosis, so EGFR has become an important target for current molecular targeted therapy (MTT). mAb drugs targeting EGFR can compete with endogenous ligands to bind to EGFR, which exhibit anti-tumor effects by inhibiting the activation of tyrosine kinases, promoting the internalization of EGFR, and other actions.

[04] There have been three anti-EGFR mAbs on the market at home and abroad, which have the characteristics of clear target, significant curative effect, small toxic and side effects, and the like compared with other chemotherapeutic drugs, and have achieved excellent clinical efficacy. The earliest anti-EGFR mAb on sale is cetuximab approved by the US FDA in 2004, which is a recombinant anti-EGFR human-mouse chimeric mAb for treating metastatic colon cancer, and this product was approved to enter the Chinese market in 2009 under a trade name of Erbitux. Panitumumab is the first recombinant anti-EGFR fully-humanized mAb, which is used for treating advanced colorectal cancer and shows lower immunogenicity and smaller potential side effects in clinical practice than chimeric mAbs such as cetuximab. Panitumumab has been approved for marketing in more than 40 countries such as European countries, America, and Japan, but China still needs to rely on imports. Imported original drugs are expensive, which limits the curing chance of most colorectal cancer patients.

[05] At present, in China, some research institutions are working in combination with some pharmaceutical companies to develop biosimilar drugs of panitumumab. However, the research and development of biosimilar drugs is different from the research and development of chemical generic drugs, which has considerable technical difficulty. Small changes in each production step will affect the expression level or activity of a final product; and the establishment and cultivation process of high-expressing cell lines, protein purification process and process control, antibody drug quality standard, and the like, all restrict the development and industrialization of biosimilar drugs.

SUMMARY

[06] In order to solve the limitations and defects in the prior art, with the original drug of panitumumab as a reference control, the present disclosure establishes a high-expressing cell line and develops a pilot-scale cultivation and purification process to obtain a recombinant anti-EGFR humanized mAb with characteristics and functions similar to that of the original drug. In the present disclosure, a stable cell line with high Vectibix expression is screened out through POOL construction, fed-batch experimental screening and plating, clone picking, HTRF, clone amplification, and clone fed-batch experiment. Through process optimization, an expression level can reach 2.05 g/L, and the SEC, pI-cIEF, deglycosylated molecular weight, and cell-based antibody activity of a product can be significantly improved.

[07] The present disclosure provides an establishment method of a cell line for preparing an anti EGFR fully-humanized mAb, where dihydrofolate reductase (DHFR)-deficient Chinese hamster ovary (CHO) cell lines CHO/DHFR- are subjected to POOL screening after undergoing transfection, and the POOL screening includes: stage 1, using an HT-free SFM4CHO medium to screen out a cell line integrated with a heavy chain (HC) gene, and using Blasticidin in the medium to screen out a cell line integrated with a light chain (LC) gene; and stage 2, adding MTX to increase copy numbers of the LC gene and the HC gene that are integrated into a genome of a cell line, thereby increasing an expression level.

[08] Preferably, at stage 1 in the POOL screening, the medium may have a GlutaMAX-I concentration of 4 mM and a Blasticidin concentration of 10 mg/l, and cells may be passaged twice a week until a cell viability is restored to more than 85%; and at stage 2, the medium may have a GlutaMAX-I concentration of 4 mM, a Blasticidin concentration of 10 mg/, and an MTX concentration of 500 nM, and cells may be passaged twice a week until a cell viability is restored to more than 90%.

[09] Preferably, during the transfection, a cell density of the DHFR-deficient CHO cell line CHO/DHFR- is adjusted to 10E5 cells/ml with a complete medium, and 10 ml of a resulting cell suspension is used for transfection; then 20 g of a linearized plasmid pCHO1.0 invitrogen, 16.7 1 of FreeStyle T M MAX Reagent, and 0.67 ml of OptiProTM SFM are added, and cells are cultivated for 6 h; and then 10 ml of CD DG44 complete medium is added, where in the CD DG44 complete medium, GlutaMAX-I has a concentration of 4 mM and PLURONIC F68 (PF68) has a concentration of 18 ml/L.

[10] Preferably, after the POOL screening is completed, clonal expansion may be conducted by 2X103 fed-batch culture; and during the 2X103 fed-batch culture, FM012 may have a concentration of 50 g/l and sugar may be supplemented to a final concentration of 10 g/l to 12 g/l in each feeding.

[11] Preferably, after the fed-batch culture, plated culture may be conducted as follows: adding cells in a centrifuge tube with a semi-solid medium at a calculated density of 300 cells/ml, thoroughly mixing and adding a resulting cell suspension to a culture plate at an amount of 2.5 ml/well, and statically cultivating the cells in an incubator with 36.5°C and 6% CO 2

.

[12] Preferably, a preparation method of the semi-solid medium used for the plated culture may include: 24 h before the plated culture, transferring 90 ml of Clone Medium CHO DHFR from °C to 2°C to 8°C in a refrigerator; adding 2 ml of GlutaMAX-I, 1 ml of Clone Detect antihuman FITC, and 2 ml of Clone XL Reagent to the ready Clone Medium CHO DHFR, and adding MTX and Blasticidin at an amount such that the MTX and Blasticidin have concentrations respectively of 500 nM and 10 mg/l in 100 ml of the semi-solid medium; and adjusting a volume of a resulting mixture to 100 ml with a host cell supernatant.

[13] Preferably, during an establishment process, cells may be cultivated under the following conditions: temperature: 36.5°C, humidity: 75% to 85%, carbon dioxide concentration: 6%, rotation radius: 2.5 cm, rotational speed: 110 rpm to 225 rpm, and volume: 20 ml to 30 ml.

[14] The present disclosure also relates to a vectibix antibody purification method, and the method includes: subjecting a vectibix antibody cell culture to the following treatments: two-stage deep filtration for clarification, affinity chromatography, low-pH incubation for virus inactivation, anion exchange chromatography, cation exchange chromatography, and concentration and dialysis.

[15] Preferably, during the two-stage deep filtration for clarification, a filter device is first rinsed at 100 L/m 2 and 600 LMH, and then equilibrated with an equilibration buffer of a volume at least 2 times a dead volume of the DOHC system at 600 LMH until a pH of an AlHC effluent is consistent with that of the equilibration buffer, where the filter device is obtained by connecting two Millipore Pod DOHC (2 x 540 cm 2 ) in parallel and connecting one Millipore Pod AlHC (540 cm 2) thereto in series; then the filtration of the vectibix antibody cell culture is started, an initial filtrate of a volume 0.5 to 0.7 times a dead volume of the entire system at an AlHC effluent end is discarded, and then the effluent end is connected to a sterilization filter in series to collect a filtrate, where a feed rate is kept at < 200 LMH and a pressure of each filter is kept at < 15 psi; after the filtration of the sample is completed, a feed solution remaining in a depth filter membrane is replaced with a buffer of a volume at least 2 times the dead volume of the entire system; and all obtainedfiltrates are combined. The equilibration buffer has a Tris-HAc concentration of 50 mM, a NaCl concentration of 150 mM, and a pH of 7.4.

[16] Preferably, during the affinity chromatography, MabSelect is used as a packing, and 5.0 x cm, CV 491 mL is used as a chromatography column; a chromatographic system is rinsed, disinfected, equilibrated, and then loaded with a combined filtrate obtained from the two-stage deep filtration for clarification; and affinity chromatography is conducted under the following conditions: flow rate: 250 cm/h; DBC < 35 mg/mL packing; retention time: > 5 min; rinse equilibration buffer: mM Na-Acetate, pH 6.0; elution equilibration buffer: 50 mM Na-Acetate, pH 3.5; and collection UV280:150-150 mAU/mm.

[17] Preferably, during the low-pH incubation for virus inactivation, an eluate obtained from the affinity chromatography is adjusted to a pH of 3.5 to 3.8, then incubated for 2 h to 4 h at room temperature for virus inactivation, and then neutralized to a pH of 5.5 to 5.8, and a neutralized product is subjected to sterilization filtration.

[18] Preferably, during the anion exchange chromatography, Super Q 650M (TOSOH) is used as a packing and 2.6 x 22.8 cm, CV 121 mL is used as a chromatography column; a product obtained from the low-pH incubation for virus inactivation is adjusted to pH 6.9, and then a chromatography system is disinfected, equilibrated, and loaded with the sample; and anion exchange chromatography is conducted under the following conditions: flow rate: 250 cm/h, DBC < 189 mg/mL packing; retention time: > 5 min; start collection UV280: 50 mAU/mm; rinse equilibration buffer: 100 mM Tris-HAc, pH 6.9; and end collection UV280: 50 mAU/mm.

[19] Preferably, during the cation exchange chromatography, Poros XS is used as a packing, and 2.6 x 25.4 cm, CV 135 mL is used as a chromatography column; a chromatographic system is disinfected, equilibrated, and then loaded with a sample; and cation exchange chromatography is conducted under the following conditions: flow rate: 250 cm/h; DBC < 65 mg/mL packing; retention time: > 5 min; rinse equilibration buffer: 50 mM Na-Acetate, pH 5.0; elution equilibration buffer: 50 mM Na-Acetate, 140 mM NaCl, pH 5.0; and collection UV280: 150-150 mAU/mm.

[20] Preferably, during the concentration and dialysis, a product is subjected to concentration and liquid exchange, where an osmotic pressure is 276 mOsm/kg, an feed flux is not more than 360 LMH, a feed end pressure is not more than 20 psi and a transmembrane pressure is not more than psi, and a concentration is adjusted to 19 mg/mL to 21 mg/mL with a dialysis buffer; and then filtration is conducted with a sterilization filter.

[21] Beneficial contributions of the present disclosure:

[22] 1. A high-efficiency and stable cell line with an expression level of 2.0 g/L to 3.0 g/L suitable for large-scale production processes is cloned and screened out for the first time, the SEC,

A pI-cIEF, deglycosylated molecular weight, and cell-based antibody activity of a product are all significantly improved, and glycosylation is also significantly improved after process optimization, which makes the industrialization of recombinant anti-EGFR humanized mAbs in China possible.

[23] 2. A four-stage purification process is independently developed, where two-stage deep filtration for clarification, affinity chromatography, low-pH incubation for virus inactivation, anion exchange chromatography, cation exchange chromatography, concentration and dialysis, and other processes are innovatively integrated. The purification process involves mild reaction conditions, is easy to be industrialized, and achieves a purification yield of > 70% and an original solution purity of > 95%, which greatly reduces the industrialization cost. The innovative use of low-pH incubation can not only inactivate pathogens without introducing exogenous residues, but also separate NGF subunits in an acidic environment, which has the advantage of "two effects with one step".

BRIEF DESCRIPTION OF DRAWINGS

[24] FIG. 1 is a logical diagram of the overall concept of the present disclosure.

[25] FIG. 2 is a schematic diagram of the cell line establishment in Example 1.

[26] FIG. 3 shows a cell viability curve for the POOL fed-batch experiment in Example 1.

[27] FIG. 4 shows a cell density curve for the POOL fed-batch experiment in Example 1.

[28] FIG. 5 shows viability curves for the fed-batch experiments of to-be-screened cell lines in Example 1.

[29] FIG. 6 shows density curves for the fed-batch experiments of to-be-screened cell lines in Example 1.

[30] FIG. 7 is a histogram illustrating expression levels for the fed-batch experiments of to-be screened cell lines in Example 1.

[31] FIG. 8 shows a cell density curve for the fed-batch experiment of a cell line screened out in Example 1.

[32] FIG. 9 shows a cell viability curve for the fed-batch experiment of a cell line screened out in Example 1.

[33] FIG. 10 shows results of HC amplification with cDNA of PCB, MCB, WCB, and EOPC cells as templates in Example 2 (M: Marker III; lane 1: cDNA, where cDNA obtained by reverse transcription of extracted total RNA is used as a template for PCR amplification; lane 2: RNA, where the original RNA without reverse transcription is used as a negative control for amplification; and lane 3: H20, where H20 is used as a negative control for PCR amplification).

[34] FIG. 11 shows results of LC amplification with cDNA of PCB, MCB, WCB, and EOPC cells as templates in Example 2 (M: Marker III; lane 1: cDNA, where cDNA obtained by reverse transcription of extracted total RNA is used as a template for PCR amplification; lane 2: RNA, where the original RNA without reverse transcription is used as a negative control for amplification; and lane 3: H2 0, where H 2 0 is used as a negative control for PCR amplification).

[35] FIG. 12 shows curves illustrating the viable cell density change over time in Example 3.

[36] FIG. 13 shows curves illustrating the cell viability change over time in Example 3.

[37] FIG. 14 shows curves illustrating the glucose concentration change over time in Example 3.

[38] FIG. 15 shows curves illustrating the lactate concentration change over time in Example 3.

[39] FIG. 16 shows curves illustrating the protein yield change over time.

[40] FIG. 17 shows affinity chromatograms in Example 5.

[41] FIG. 18 shows anion exchange chromatograms in Example 5.

[42] FIG. 19 shows cation exchange chromatograms in Example 5.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[43] In order to enable those skilled in the art to better understand the technical solutions of the present disclosure, the establishment method of a cell line for preparing an anti-EGFR fully humanized mAb provided by the present disclosure will be described in detail below in conjunction with examples.

[44] As shown in FIG. 1 and FIG. 2, the present disclosure uses the original drug Vectibix (panitumumab) as a reference product to develop a biosimilar drug with the same amino acid sequence as Vectibix. On the basis of the preliminary small-scale process, methods and conditions are explored and optimized for the 200 L pilot-scale process to determine key process parameters and form a complete quality control system and standard, which lays a foundation for the further large-scale industrialization of the recombinant anti-EGFR fully-humanized mAb. The present disclosure adopts an independent innovative process to investigate and develop a pilot-scale production process of the recombinant anti-EGFR humanized mAb, which is intended to make breakthroughs in the three key technologies of efficient and stable cell line establishment, cell cultivation, and purification. Through technological transformation and process innovation, the present disclosure makes a product quality meet or exceed a quality of the foreign original drug while greatly reducing a preparation cost, such that more colorectal cancer patients can be cured.

[45] The control standards used in the following examples of the present disclosure are all commercially-available Vectibix 400 mg/20 ml (intravenous infusion solution of panitumumab, Amgen).

[46] Cell line establishment

[47] The cell line establishment includes Examples 1 and 2. The establishment of a stable and efficient cell line is the key to the domestic industrialization of recombinant anti-EGFR fully humanized mAbs. In the examples, 50 primary cell banks (PCBs), 100 master cell banks (MCBs), and 200 work cell banks (WCBs) are prepared by establishing CHO/DHFR- engineered cell lines for producing recombinant anti-EGFR humanized mAb (vectibix) drugs to screen out a cell line with high Vectibix expression suitable for large-scale production processes. Through process optimization, an expression level is above 2.0 g/L and the oldest cell expression level is not less than 70%, which is suitable for scale-up production; and the SEC, pI-cEF, deglycosylated molecular weight, and cell-based antibody activity of a product and the glycosylation all reach the level of the original drug.

[48] Example 1

[49] Transfection experiment:

[50] Experimental materials:

[51] Host cell: DG44 (DHFR-deficient CHO cell line CHO/DHFR-, ATCC)

[52] Transfection reagent: Freestyle Max Reagent (Invitrogen 16447-100)

[53] OPTI PRO SFM(GIBCO 12309-019)

[54] Linearized plasmid: pCHO1.0 invitrogen

[55] DG44 complete medium: CD DG44 (Invitrogen 12610-010) with

[56] 4 mM GlutaMAX-I (Invitrogen 35050-061) and

[57] 18 ml/L PLURONIC F68(PF68) (Invitrogen 24040-032)

[58] Consumables and related instruments

[59] Shaking flask: CORNING 125 ml Erlenmeyer Flask, Item No.: 431143;

[60] Shaker: INFORS HT, Model: Multitron II

[61] Parameter setting: 110 rpm, 36.5°C, 6% CO 2 , humidity: 75%;

[62] Cell counter: BECKMAN COULTER, model: VI-CELL XR

[63] Biosafety cabinet: Shanghai Shangjing, Model: BSC- II A2

[64] Transfection experiment steps:

[65] 1. 24 h before transfection, spare DG44 cells to be transfected were subcultivated at a density of 8E5/ml with a CD DG44 complete medium.

[66] 2. On the day of transfection, the spare cells were subjected to cell counting and cell viability assay.

[67] 3. A CD DG44 complete medium was used to adjust a cell density to 10E5 cells/ml, 10 ml of a resulting cell suspension was used for transfection, and a 125 ml shaking flask was adopted.

[68] 4. 20 g of a plasmid (pCHO1.0 invitrogen) and 16.7 1 of FreeStyle T M MAX Reagent were added to 0.67 ml of OptiProTM SFM, separately, and a resulting mixture was thoroughly mixed.

[69] 5. The mixture of plasmid and transfection reagent was incubated at room temperature for min and then added to the previously prepared cells, and the cells were cultivated in a shaker.

[70] 6. 6 h after the cultivation, 10 ml of DG44 complete medium was added, and the cells were further cultivated.

[71] POOL screening experiment:

[72] Consumables and related instruments

[73] Shaking flask: CORNING 125 ml Erlenmeyer Flask, Item No.: 431143

[74] Shaker: INFORS HT, Model: Multitron II

[75] Parameter setting: 110 rpm, 36.5°C, 6% CO 2 , humidity: 75%

[76] Cell counter: BECKMAN COULTER, model: VI-CELL XR

[77] Biosafety cabinet: Shanghai Shangjing, Model: BSC- II A2

[78] Centrifuge: EPPENDORF, model: Centrifuge 5810R

[79] The medium information for the fed-batch experiment was shown in Table 1:

[80] Table 1

Medium name Brand Item No. SFM4CHO Hyclone SH30518.01 Glutamine Sigma G8540 MTX Sigma A6770-1 Blasticidin Invitrogen Al 1139-03

[81] POOL screening experiment steps

[82] Step I. After the transfection, an HT-free SFM4CHO medium was used to screen out a cell line integrated with an HC gene, and a cell line integrated with an LC gene was screened out by Blasticidin in the medium. Step II. MTX was added to increase copy numbers of the LC gene and HC gene that were integrated into a genome of a cell line, thus increasing an expression level. Through the two-step pressure screening, a high-expressing stable cell line simultaneously integrated with light and HC sequences could be screened out.

[83] Step 1:

[84] Basic medium: SFM4CHO

[85] which included 4 mM GlutaMAX-I and 10 mg/l Blasticidin.

[86] Cell passage: Cells were passaged twice a week in 125 ml shaking flasks until a cell viability was restored to more than 85%. Cells were cryopreserved twice, with 2 tubes each time.

[87] Step 2:

[88] Basic medium: SFM4CHO

[89] which included 4 mM GlutaMAX-I, 10 mg/l Blasticidin, and 500 nM MTX.

[90] Cell passage: Cells were passaged twice a week in 125 ml shaking flasks until a cell viability was restored to more than 90%. Cells were cryopreserved twice, with 2 tubes each time.

[91] POOL fed-batch

[92] Consumables and related instruments

[93] Shaking flask: CORNING 125 ml Erlenmeyer Flask, Item No.: 431143

[94] Shaker: INFORS HT, Model: Multitron II

[95] Parameter setting: 110 rpm, 36.5°C, 6% CO 2 , humidity: 75%

[96] Cell counter: BECKMAN COULTER, model: VI-CELL XR

[97] Biosafety cabinet: Shanghai Shangjing, Model: BSC- II A2

[98] The medium information for the fed-batch experiment was shown in Table 2 below:

[99] Table 2 Medium name Brand Item No. SFM4 CHO Hyclone SH30518.01 Cell Boost 5 (CB5) Hyclone SH30865.03 FMO12 Sheffield 5X00483 2X103 Duoning F103-201 GlutaMAX-I GIBCO 35050-061 Glucose Sigma 49139

[100] Experimental scheme:

[101] When the cell viability was restored to more than 90%, the fed-batch experiment was conducted in a 125 ml shaking flask.

[102] POOL name: JN-XBK(10 mg/l Blasticidin+ 500 nM MTX)

[103] The cells were inoculated at 5E5 cells/ml and cultivated for 4 d in the medium SFM4CHO. The cells, after growing for 4 d, were diluted at 1:5 with a fed-batch basic medium (90% SFM4CHO + 10% CB5).

[104] Fed-batch experimental scheme:

[105] Feeding: 2X103; 50 g/l FMO12 in 2X103.

[106] Sugar was supplemented to a final concentration of 10 g/l to 12 g/l in each feeding.

[107] Experimental conditions were shown in Table 3:

[108] Table 3

Sample D3 D6 D8 D1O 5% (50g/l 10 bo(50g/l FMO12 in 10%o(50g/l FMO12 in 5% (50g/l JN-XBK FMO12in3 in 10% 2X103M FMO12 in 2X103) 2X103) Cultivation conditions Temperature Carbon Equipment Rotational Volume(ml) (0 C0 Humidity(0%) dioxide(0%) rotation radius speed (rpm) Voue(l (cm) 36.5 75 6 2.5 110 30

[109] Results of the fed-batch experiment were shown in FIG. 2 and FIG. 3. Supernatants collected in the Pool fed-batch experiment were subjected to titer detection by HPLC-Protein A, and results showed that a titer on day 13 of JN-XBK was 1.67 g/L.

[110] Semi-solid medium plating:

[111] Equipment and consumables:

[112] 6-well plate: Greiner Bio-One Cell Star, Item No.: 657185

[113] Cell counter: BECKMAN COULTER, model: VI-CELL XR

[114] Carbon dioxide static incubator: THERMO SCIENTIFIC, Model: HERA ce11240i

[115] Thermostat water bath: Thermo, Microprocessor controlled 280, 475524

[116] Biosafety cabinet: Shanghai Shangjing, Model: BSC- II A2

[117] The medium and reagents were shown in Table 4:

[118] Table 4

Composition Supplier Item No. CloneMedium CHO DHFR Genetix K8712 Clone XL Reagent Genetix K8521 GlutaMAX-I Gibco 35050-061 MTX Sigma A6770-1 Blasticidin Invitrogen Al 1139-03 CloneDetect anti-human FITC Genetix K8200

[119] Preparation steps of the semi-solid medium:

[120] 24 h before plating, a 90 ml bottle of CloneMedium CHO DHFR was transferred from °C to 2°C to 8°C in a refrigerator for later use.

[121] 2 ml of GlutaMAX-I, 1 ml of Clone Detect antihuman FITC, and 2 ml of Clone XL Reagent were added to the prepared CloneMedium CHO DHFR.

[122] MTX and Blasticidin were added to the prepared CloneMedium CHO DHFR such that the MTX and Blasticidin had concentrations respectively of 500 nM and 10 mg/l in 100 ml of the semi solid medium.

[123] A volume of a resulting mixture was adjusted to 100 ml with a host cell supernatant.

[124] Plating steps:

[125] 1. Cell count: cultivated Pool JN-XBK was collected and counted with Vicell-XR for later use.

[126] 2. The prepared uniform semi-solid medium was taken and added to two 50 ml sterile centrifuge tubes, each with 10 ml.

[127] 3. The counted cells were added to the centrifuge tubes in step 2 at a cell density of 300 cells/ml, and resulting mixtures were thoroughly mixed.

[128] 4. A pipette was used to transfer the prepared cell suspension to be plated into a 6-well plate at 2.5 ml/well.

[129] 5. After the plating was completed, the cells were statically cultivated in an incubator with 36.5°C and 6% C02.

[130] Example 2

[131] Equipment and consumables

[132] Thermostat water bath: Julabo, model: TW20

[133] Biosafety cabinet: Shanghai Shangjing, Model: BSC- II A2

[134] Carbon dioxide static incubator: THERMO SCIENTIFIC, Model: HERA ce11240i

[135] Cell counter: BECKMAN COULTER, model: VI-CELL XR

[136] Centrifuge: EPPENDORF, model: Centrifuge 5810R

[137] ClonePix GENETIX, model: Clonepix FL

[138] 96-well plate (greiner bio-one CELLSTAR, Item No.: 655185)

[139] 24-well plate (greiner bio-one CELLSTAR, Item No.: 662102)

[140] 6-well plate (greiner bio-one CELLSTAR, Item No.: 657185)

[141] Shaker: KUHNER, model: Climo-Shaker ISF1-XC

[142] Parameter setting: 225 rpm, 36.5°C, 6% C0 2 , humidity: 85%

[143] Shaking tube: TPP TUBESPIN BIOREACTOR 50, Item No.: 87050

[144] The medium and reagents were shown in Table 5 below:

[145] Table 5

Medium name Brand Item No. SFM4 ADCF Hyclone SH3A2156.01 Glutamine Sigma G8540 MTX Sigma A6770-1 Blasticidin Invitrogen Al1139-03 Cell Boost5 (CB5) Hyclone SH30865.03 FM012 Sheffield 5X00483 2X103A Duoning F103-201.A Glucose Sigma 49139 BR7 Duoning F008-007

[146] Clone picking

[147] The growth status of clones was observed, and the clones were picked after growing into a uniform spherical shape.

[148] 1. A 96-well plate was taken and each well was added with 100 1 of fresh medium SFM4ADCF (with 4 mM Glutamine, 1 g/L PF68, 10 mg/l Blasticidin, and 500 nM MTX).

[149] 2. Pool JN-XBK clones were picked and transferred to the 96-well plate with ClonePix (SFM4 ADCF + 10 mg/l Blasticidin + 500 nM MTX).

[150] 3. The 96-well plate was transferred to a 36.5°C and 6% CO 2 incubator for static cultivation.

[151] HTRF clone screening and amplification

[152] Medium: SFM4ADCF (with 4 mM Glutamine, 1 g/L PF68, 10 mg/l Blasticidin, and 500 nM MTX)

[153] 1. On day 7 after the clone picking, 100 [ of fresh medium was added to the 96-well plate.

[154] 2. A supernatant was collected for HTRF assay, clones with high expression were selected and transferred to a 24-well plate, and 400 1 of fresh medium was added to each well.

[155] 3. After cultivated for 3 d to 4 d, the clones in the 24-well plate were transferred to a 6-well plate, and 2 ml of fresh medium was added to each well.

[156] 4. After cultivated for 3 d to 4 d, the clones in the 6-well plate were transferred to a shaking tube, and 8 ml of fresh medium was added for each clone.

[157] 5. Two clones with high HTRF were picked for plating according to the above operation, and subclones were picked, screened, and amplified.

[158] Fed-batch clone screening

[159] Cells were passaged twice in shaking tubes, and then inoculated at 3E5 cells/ml and cultivated for 4 d in the medium SFM4ADCF. After 4 d of growth, the cells were diluted at 1:5 with a fed-batch basic medium (90% SFM4ADCF + 10% CB5), and the fed-batch experiment was conducted in a 50 ml shaking tube. Subclones of Pool JN-XBK were named JK-I, JK-II, and JK-III, respectively. Selected Top3 clones were JK-I, JK-II, and JK-III.

[160] The fed-batch experimental scheme was shown in Tables 6 and 7 below.

[161] Feeding: 30g/lFM012in2X103A;BR7.

[162] Sugar was supplemented to a final concentration of 10 g/l to 12 g/ in each feeding.

[163] Table 6

Sample D4 D7 D9 D1I 8%FM012 in 10%FM012 in 10%FM012 in 10%FM012 in JK-I 2X103A+0.8%B 2X103A+1%BR 2X103A+1%B 2X103A+1%BR R7 7 R7 7 8%FM012 in 10%FM012 in 10%FM012 in 10%FM012 in JK-II 2X103A+0.8%B 2X103A+1%BR 2X103A+1%B 2X103A+1%BR R7 7 R7 7 8%FM012 in 10%FM012 in 10%FM012 in 10%FM012 in JK-III 2X103A+0.8%B 2X103A+1%BR 2X103A+1%B 2X103A+1%BR R7 7 R7 7

[164] Table 7

Cultivation conditions: Temperature Humidity Carbon dioxide Equipment rotation Rotational Volume (0 C) (%) (%) radius (cm) speed (rpm) (ml) 36.5 85 6 2.5 225 20

[165] Results of the fed-batch clone screening experiment were shown in FIG. 4 to FIG. 5. Titer

All% results detected by Protein A-HPLC in the fed-batch clone screening experiment were shown in FIG. 6, and the highest JK-I yield was 1.82 g/, which was selected for the further shaking flask optimization and quality analysis and the upstream process development.

[166] Shaking flask optimization experiment and quality analysis of a selected clone

[167] Shaking flask optimization experiment of a selected clone

[168] Clone name: JK-I

[169] The cells were inoculated at 3E5 cells/ml and cultivated for 4 d in the medium SFM4ADCF. After 4 d of growth, the cells were diluted at 1:5 with a self-prepared fed-batch basic medium. The fed-batch experiment was conducted in a 50 ml shaking tube.

[170] The fed-batch experimental scheme was shown in Tables 8 and 9.

[171] Medium: SFM4 ADCF + supplements

[172] Feeding: 30 g/L FM012 in 2X103A; BR7.

[173] Sugar was supplemented to a final concentration of 10 g/l to 12 g/ in each feeding.

[174] Table 8

Sample D3 D7 D9 D1I 5%FM012 in 10%FMO12 in 10%FM12 in 10%FM012 in JK-I 2X103A+0.5% 2X103A+1%B 2X103A+1i%BR 7 2X103A+1%B BR7 R7 R7

[175] Table 9

Cultivation conditions: Temperature Humidity Carbon dioxide Equipment rotation Rotational Volume (°C) (%) (%) radius (cm) speed (rpm) (ml) 35 85 6 2.5 225 20

[176] Experimental results were shown in FIG. 7 and FIG. 8. The titer assay was conducted by protein A-HPLC after the selected clone was optimized by the fed-batch experiment, and results showed that a yield on day 14 was 2.05 g/l.

[177] Quality analysis results and optimization of a selected clone

[178] A cell supernatant of the fed-batch experiment was purified by Protein A in one step and a sample was collected for quality analysis.

[179] Table 10 Purity SEC-HPLC

Test Result SampleID Main peak% HMW peak% LMW peak% INV (1033112) 97.4 2.6 ND JK-I 98.5 1.5 ND

[180] Purity SEC-HPLC analysis results were close to that of the original drug (Vectibix), and the indexes met the requirements.

[181] Table 11 pI-clEF

Test Result Sample ID pI Main peak % Acidic Basic peak

% _________________peak00

INV (1033112) 7.0 43.4 12.5 44.1 JK-I 7.0 54.4 17.4 28.3

[182] According to pI-clEF test, the antibody had an isoelectric point consistent with that of the original drug, which met the requirements.

[183] Glycan

[184] Before optimization: Table 12

Test Result Sample ID GO GOF Man-5 G2 G2F Others ______________ (0) (0) (0) (0) (0) (0)

INV (1033112) 1.0 37.8 3.8 0.1 8.1 7.4 JK-I 0.2 66.3 0.6 NA 2.6 4.4

[185] After optimization: Table 13

Test Result SampleID GOF GIF G2F INV (1033112) 39.4% 45.3% 15.3% JK-I 41.0% 45.1% 13.9%

[186] A glycosylation test report showed that GOF, GIF, and G2F were basically the same as that of the original drug, which met the requirements.

[187] Table 14 Cell Based Assay

Sample ID Test Result JK-I 88%

[188] According to cell-based antibody activity assay, the antibody had an activity close to that of the original drug, which met the requirements.

[189] Table 15 Deglycosylated mass

Sample ID Theoretical Mass Analyte Mass Error (Da) (Da) Mass (Da) JK-I 144035.7 144041.5 5.8

1 A

[190] According to deglycosylated mass analysis, the antibody had a deglycosylated molecular weight consistent with a theoretical value.

[191] In the present disclosure, a stable cell line JK-I with high Vectibix expression was screened out through POOL construction, fed-batch experimental screening and plating, clone picking, HTRF, clone amplification, and clone fed-batch experiment. Through process optimization, an expression level reached 2.05 g/L, and the SEC, pI-cIEF, deglycosylated molecular weight, and cell-based antibody activity of a product all were significantly improved.

[192] Example 3 Cell line stability test

[193] The genetic stability of a cell line was the basis for subsequent stable production processes and product quality. With a molecular biotechnology, the genetic stability was investigated for cells in the three cell banks of PCB, MCB, and WCB in the project, including HC and LC DNA sequence stability and copy number stability. The project was intended to conduct cell line stability research for no less than 12 weeks, including evaluation of cell growth stability, product expression stability, and molecular level cell line stability.

[194] With a molecular biotechnology, the genetic stability was investigated for cells in the three cell banks of PCB, MCB, and WCB in Example 1 and end of production cells (EOPCs) in pilot scale production, including HC and LC DNA sequence stability and copy number stability. The copy number stability was determined as follows: genomic DNA (gDNA) was extracted separately from cells in PCB, MCB, and WCB and from EOPCs in pilot-scale production (cell number: about 1 x 107), and a qPCR amplification result of an HC or LC gene was compared with that of an internal reference gene p2-microglobulin (B2M) to determine copy number variations (CNVs) of HC and LC genes of cells in PCB, MCB, and WCB and of EOPCs in pilot-scale production. The stability of HC and LC DNA sequences was investigated as follows: total RNA was extracted from PCB, MCB, and WCB cells and EOPCs (total cell number: about 1 x 107), separately; with the total RNA as a template, reverse transcription was conducted to obtain cDNA, which included HC and LC cDNA; then with the cDNA as a template, synthesized primers were used to amplify HC and LC genes, separately; and PCR products were finally sequenced to determine whether the cell bank cells and EOPCs had mutations at the genetic level, thereby evaluating the stability of the cell banks.

[195] Test results showed that, in the PCB, MCB, and WCB cells and the EOPCs in pilot-scale production of Example 1, there was no significant change in the copy numbers of HC and LC genes; and a target product was amplified at the cDNA level, and a PCR product was sent to a sequencing company for sequencing to determine whether the cell bank cells and EOPCs had mutations at the genetic level.

[196] Experimental instruments

[197] 7500 Fast Real-Time PCR System (Applied Biosystems, Model#7500)

[198] BioSafety Cabinet (AIRTECH, Model#BSC-130011 A2)

[199] Experimental consumables

[200] Dneasy Blood & Tissue Kit(250) (QIAGEN, Cat. #69506)

[201] TaqMan Universal Master Mix II with (AB,Cat.#4426710)

[202] UNG

[203] RNeasy mini kit (QIAGEN, Cat. #74104)

[204] Superscript III First-Strand Synthesis (Invitrogen, Cat. #No18080-051)

[205] System for RT-PCR kit

[206] PrimeSTAR DNA polymerase (TaKaRa, Cat. #DR044A)

[207] Experimental samples

[208] One tube of each of JK-I-PCB, JK-I-MCB, and JK-I-WCB in Example 1 was thawed, then cultivated, and centrifuged to obtain a cell precipitate (1E7) for later use. The names, batch numbers, and sources of the three cell banks and EOPCs were shown in Table 16.

[209] Table 16 Names, batch numbers, and sources of experimental samples

Cell bank Sample name or Batch No. Source J-I-PCB JK-I-PCB-13 J-I-MCB JK-I-MCB Example 1 J-I-WCB JK-I-WCB JNY-EOPC JNY-S128X01-EOPC Cell Culture Process Development

[210] The copy number stability was assayed for HC and LC genes in PCB, MCB, and WCB cells and EOPCs of Example 1.

[211] 1. gDNA extraction

[212] gDNA was extracted from PCB, MCB, and WCB cells and EOPCs of JK-I in Example 1, and specific steps could be seen in the Dneasy Blood & Tissue Kit instruction manual.

[213] 2. Design of primers and probes

[214] Combinations of primers and probes that could specifically bind to the HC, LC, or internal reference gene were designed, as shown in Table 17.

[215] Table 17

Category No. Primer sequence SEQ ID No.1 5'-TGGAGTGGGAGAGCAATGG-3' HC Primer SEQ ID No.2 5'-GCATGGGAGGTGTGGTCTTG-3' MGB probe SEQ ID No.3 5'-AGCCGGAGAACAACT-3' SEQ ID No.4 5'-CACCCTGACGCTGAGCAAA-3' LC Primer SEQ ID No.5 5'-TGACTTCGCAGGCGTAGACTT-3' MGB probe SEQ ID No.6 5'-CAGACTACGAGAAACAC-3' Internal Primer SEQ ID No.7 5'-TTGGGCCCTTGGTGCTT-3'

A /' reference SEQ ID No.8 5'-AAACCGAAAGTAGATGCTTGGAA-3' B2M TAMRA probe SEQ ID No.9 5'-CTTCCTTGTTGGCCCGCTGCC-3'

[216] 3. qPCR settings

[217] With gDNA of PCB, MCB, and WCB cells and EOPCs at three concentrations of 50 ng/5 dl, 10 ng/5 d, and 5 ng/5 [ as substrates, 25 1 reaction systems were prepared according to instructions of TaqMan Universal Master Mix II with UNG. ABI 7500 Fast Real Time System was set according to the Comparative-Ct (AACt) method, and then qPCR was conducted.

[218] The stability was assayed for HC and LC gene sequences in PCB, MCB, and WCB cells and EOPCs of Example 1.

[219] 1. Extraction of total RNA from cell bank cells

[220] Total RNA was extracted from PCB, MCB, and WCB cells and EOPCs of JK-I in Example 1, and specific steps could be seen in the RNeasy mini kit instruction manual.

[221] 2. Synthesis of a first strand of cDNA for an antibody gene

[222] OligodT was used to amplify a cDNA fragment by reverse transcription, and experimental steps could refer to the operation process of First Script III First-Strand Synthesis System for RT PCR kit.

[223] 3. Amplification of HC and LC sequences

[224] With cDNA obtained by reverse transcription as a template, the HC and LC fragments for the antibody were amplified using primers shown in Table 18, and PCR amplification products were sent to a sequencing company for sequencing.

[225] Table 18 Amplification primers for HC and LC fragments in cDNA and sequences thereof

Primer No. Nucleotide sequence HC SEQ ID No.10 5'-CATTGACGCAAATGGGCGGTAG-3' SEQ ID No. 11 5'-ATATGGTGGAAAAT AACAT A-3' LC SEQ ID No. 12 5'-ACGCTGTTTTGACCTCCATAGAA-3' SEQ ID No. 13 5' -GTCATAGCGCGGGTTCCTTCCG-3'

[226] Different amplification reactions for HC and LC genes in PCB, MCB, and WCB cells and EOPCs

[227] Ct value differences calculated for the HC and LC genes in cell bank cells and EOPCs of JK-I in Example 1 relative to the internal reference gene B2M were summarized in the following table. The average value and standard deviation of ACt values calculated at different sample concentrations were also listed in Table 19 below.

[228] Table 19 Calculation of ACt (HC-Ref) and ACt (LC-Ref)

J-I PCB J-I MCB ACt HC-B2M LC-B2M HC-B2M LC-B2M 50 ng -10.96 -10.79 -10.67 -10.72 10 ng -10.94 -10.76 -10.91 -10.77 5 ng -10.92 -10.85 -10.54 -10.64 Mean (ACt) -10.94 -10.80 -10.71 -10.71 StdDev (ACt) 0.02 0.04 0.19 0.07 J-I WCB J-I EOPC ACt HC-B2M LC-B2M HC-B2M LC-B2M 50 ng -10.64 -10.66 -10.95 -11.00 10 ng -10.63 -10.89 -10.92 -10.86 5 ng -10.68 -10.60 -11.00 -11.09 Mean(ACt) -10.65 -10.71 -10.96 -10.98 StdDev(ACt) 0.03 0.16 0.04 0.11

[229] CNVs for HC and LC genes in PCB, MCB, and WCB cells and EOPCs

[230] The relative copy numbers of HC and LC genes in PCB of JK-I in Example 1 were standardized to 1 relative to the internal reference gene B2M, and the CNVs of HC and LC genes in MCB and WCB cells and EOPCs relative to that in PCB were summarized in Table 20.

[231] Table 20 CNVs of HC and LC genes in PCB, MCB, and WCB cells and EOPCs of JK-I in Example 1

HC PCB MCB WCB EOPC -AACt 0.00 -0.23 -0.29 0.02 2-AACt 1.00 0.85 0.82 1.01 LC PCB MCB WCB EOPC -AACt 0.00 -0.09 -0.09 0.18 2-AACt 1.00 0.94 0.94 1.14

[232] According to analysis by the Comparative-Ct (AACt) method, the copy number of HC gene in MCB was reduced by 15% compared to that in PCB, the copy number of HC gene in WCB is reduced by 18% compared to that in PCB, and the copy number of HC gene in EOPC was increased by 1% compared to that in PCB; and the copy number of LC gene in MCB was reduced by 6% compared to that in PCB, the copy number of LC gene in WCB was reduced by 6% compared to that in PCB, and the copy number of LC gene in EOPC was increased by 14% compared to that in PCB. The CNVs of the HC and LC genes in MCB and WCB cells and EOPCs of Example 1 were all within an allowable error range for the experiment.

[233] Stability assay results for HC and LC gene sequences in PCB, MCB, and WCB cells and EOPCs of Example 1

[234] With the cDNA obtained by reverse transcription as a template, the HC (about 1,395 bp) and LC fragments (711 bp) for the antibody were amplified, separately, PCR amplification products

In0 were subjected to agarose gel electrophoresis, and electrophoresis results were shown in FIG. 10 and FIG. 11.

[235] Obtained sequencing results of HC and LC genes in PCB, MCB, and WCB cells and EOPCs were compared with a theoretical sequence by the Vector NTI software, and all sequencing results were 100% consistent with the theoretical sequence.

[236] Result analysis by the Comparative-Ct (AACt) method in qPCR showed that the copy numbers of HC and LC genes in PCB, MCB, and WCB cells and EOPCs of Example 1 were relatively stable. Sequence alignment analysis showed that the HC and LC cDNA in PCB, MCB, and WCB cells and EOPCs of Example 1 remained relatively stable at the genetic level, and there was no gene mutation, base deletion, or base increase in the HC and LC sequences. In summary, the PCB, MCB, and WCB cells and EOPCs in Example 1 had prominent genetic stability.

[237] Example 4 Optimization of pilot-scale cell cultivation process

[238] After a monoclonal cell line was obtained, cultivation conditions were optimized to achieve a high expression level and meet the specified product quality. The depletion of nutrients and the accumulation of lots of metabolic by-products during a cell cultivation process are often the main factors that limit the cell growth density and affect the cultivation process. Especially in a fed-batch cultivation process, the continuous consumption of nutrients often becomes a restricting factor that affects the cell density, cell viability, and cell expression product yield.

[239] In this example, on the basis of a small-scale cultivation process, the scale was expanded and different parameter control ranges were optimized to design a product quality. The cell growth and mAb production kinetics were investigated, and the influence of physical parameters (including temperature, gas flow, and stirring speed), chemical parameters (including dissolved oxygen and carbon dioxide, pH, osmotic pressure, and oxidation-reduction potential (ORP)), metabolite levels (including basic materials in medium), amino acids and waste (by-products), feed time, foam control, and the like on cell growth and protein yield was analyzed to determine the optimal environmental conditions for pilot-scale cell cultivation.

[240] The medium had been determined through 2 rounds of reactor experiments and 3 rounds of shaking flask experiments early; the following were also verified: the optimal cultivation temperature in the whole process: 35°C, the optimal feeding: 2 x 104 (including 50 g/L FM12), and feeding time: day 4, day 7, day 9, and day 11; and through the FG01 fed-batch experiment, it was also determined that 0.28%, 0.4%, 0.4%, and 0.4% were fed on day 4, day 7, day 9, and day 11, respectively. Finally, the small-scale cell cultivation process was determined. In the scale-up experiment of this example, the fed-batch cultivation method was used to conduct the pilot-scale cultivation in a 200 L reactor. The cell viability, cell metabolism, protein yield, etc. were observed to optimize the pilot-scale process, the influence of cultivation volume, inoculation density, reactor

A r% parameter control, cultivation conditions, feeding strategy (day 3, day 6, day 9, and day 11), and feeding concentration and ratio on cell growth and protein yield was investigated, and three batches of products were tested and compared with the original product, thus finally determining key process parameters and the pilot-scale process.

[241] The cell line used in the cell cultivation technology research in this report was the JK-I of Example 1, which was the cell line determined by the cell line development through the clone screening experiment, hereinafter referred to as "JK-I clone".

[242] 3 batches of 15 L reactor experiments were mainly introduced below to verify the process obtained by optimization of the small-scale process. Experimental data showed that the cell growth and metabolism parameters in the three batches were basically the same, and protein yields were 2.35 g/L, 2.39 g/L, and 2.45 g/L at harvest, respectively; and acid peak areas in the CEX test results were all lower than 13.3%, and GOF in the glycoform distribution results was 47% to 48%. Results of 3 batches of 15 L experiments showed excellent repeatability, which verified the stability of the process determined from the small-scale process; and according to this process, 3 batches of 200 L production were conducted.

[243] Table 21 Explanation of technical terms

PDL Population Double Level CLD Cell line development PAS Protein Analysis VCC Viable Cell Concentration VIA Viability DO Dissolved Oxygen INV Innovator CEX Cation-Exchange Chromatography HPLC High Performance Liquid Chromatography WCB Work Cell Bank

[244] Table 22 Equipment

Equipment Equipment name manufacturer Model Bioreactor controller applikon applikon ez- Control NOVA biochemical nova BioProfile@ FELX detector Blood gas analyzer SIMENS RAPIDLAB@ 248 Cell counter BECKMAN Vi-cell XR Ultra-low temperature freezer Haier DW-86L-626 Ultra-low temperature freezer NBS U570 Electronic balance sartorius BSA5201-CW Electronic balance OHRUS Scout Pro SPS4001F

Platform scale OHRUS ExplorerPro EP32001 Platform scale sartorius Midrics Tube sealing mahine sartorius 16360-P3 (1.6/2.4mm) Tube connecting mahine TERUMO SCD@IIB Shaker INFORS Multitron Shaker INFORS Multitron Pro Shaker Kuhnner ISF1-XC Biosafety cabinet AIRTECH BSC-1000 II A2 Biosafety cabinet AIRTECH BSC-1300 II A2 Biosafety cabinet AIRTECH BSC-1600 II A2 Autoclave HIRAYAMA HV-85L Autoclave BMT SP 969-1 Centrifuge eppendorf 5418 Centrifuge BECKMAN Allegra X-12R Integrity tester sartorius sartocheck 3

+

[245] Table 23 Consumables

Consumable name Brand Item No. 125 ml shaking flask Corning 431143 250 ml shaking flask Corning 431144 1000 ml shaking flask coming 431147 2 ml pipette Corning 4486 ml pipette Corning 4487 ml pipette Corning 4488 ml pipette Corning 4489 ml pipette Corning 7017 100 ml pipette Corning 7000 1.5ml centrifuge tube Axygen Mct-150-C ml centrifuge tube JET CFT-011-150 200 ul pipette tip Axygen T-200-Y 1000 ul pipette tip Axygen T-1000-B ml Sterile screw-top syringe Laien NA Counting cup Beckman 383721 0.2 um syringe filter Millipore SLGP033RB Air filter (small) Sartorius 17575-Q Air filter (large) Sartorius 17805-G Sterilization syringe nylon Sartorius 17845-ACK membrane filter Silicone tube No. 14 Saint-Gobain FA2918 Silicone tube No. 16 Saint-Gobain FA2543 Thermoplastic tube No. 5 Saint-Gobain 374-125-2

[246] Table 24 Medium composition

Medium Brand Item No. 2x104 Duoning Biotechnology F104-001 CB5 Hyclone SH30865.03

SFM4 ADCF Hyclone SH3A2156.01 FM012 Sheffield 5X00483 L-Glutamine Sigma G8540 Sodium bicarbonate Sigma S5761 Sodium carbonate Sigma S7795 Glucose Sigma G7021 PF68 Spectrum P1169 MTX Sigma M9929 BS (Blasticidin S HCl) Gibco Al1139-03 Medium Brand Item No. 2x104 Duoning Biotechnology F104-001 CB5 Hyclone SH30865.03 SFM4 ADCF Hyclone SH3A2156.01 FM012 Sheffield 5X00483 L-Glutamine Sigma G8540 Sodium bicarbonate Sigma S5761 Sodium carbonate Sigma S7795 Glucose Sigma G7021 PF68 Spectrum P1169 MTX Sigma M9929 BS (Blasticidin S HCl) Gibco Al1139-03

[247] The general parameter settings of a shaker involved in this experiment were shown in the table below. If it was not a general setting, the description of a corresponding part was referred for details.

[248] Table 25 General parameter settings of a shaker

Shaker parameter Setting Unit CO 2 6 %

Temperature 36.5 0C

Humidity 80 %

Shaker rotational speed 125 RPM

[249] A sample collection plan used in the fed-batch cultivation stage was shown in the table below. If it was not a general sampling plan, the description of a corresponding section was referred for details.

[250] Table 26 Sample collection and retention plan

Sample retention Daily sampling Sample retention to determine content protein yield Sample retention time Day 0 to 15 Day 12/14/15 Quantity specification 15 mL to 20 mL 0.5 mL x 2 14,000 g, 5 min, room temperature Treatment method NA When the test is completed, a sample is sent to the PAS department for detecting a protein expression level by HPLC. * Off-line detection of pH: BGA * Detection of viable cell density and cell viability by Vi-cell Detection items Detection of metabolic biochemical indexes (glucose, Proteinyield lactic acid, etc.): Nova-flex * Detection of osmotic pressure: Nova-flex Retention method The remaining is discarded Cryopreservation at -80°C Remarks

[251] 15 L reactor process verification experiment

[252] Preliminary experimental results

[253] Through 2 rounds of reactor experiments and 3 rounds of shaking flask experiments, the following were verified: the optimal cultivation temperature in the whole process: 35°C, the optimal feeding: 2 x 104 (including 50 g/L FMO12), and feeding time: day 4, day 7, day 9, and day 11; and through the FG01 fed-batch experiment, it was also determined that 0.28%, 0.4%, 0.4%, and 0.4% were fed on day 4, day 7, day 9, and day 11, respectively. Finally, a cell cultivation process was determined. In this round of experiments, three batches of process stability verification were conducted on a 15 L reactor scale.

[254] Seed cultivation information

[255] Cell source

[256] Table 27 Clone information table

Cultivation batch No. Clone name Cell bank Cell source PDL in cultivation 14 15L (PD1-UA1) JK-I WCB CLD 69.02 2 nd 15L(PD2-UA1) JK-I WCB CLD 83.25 3 rd 15L(PD3-UA8) JK-I WCB CLD 87.22

[257] Passage and expansion cultivation information:

[258] Table 28 Seed passage and expansion medium information

Cultivation stage Seed passage N-2 N-1 Basic medium SFM4 SFM4: CB5 (volume ratio: 9:1) Selected pressure and 500nM MTX+10mg/L BS No concentration

[259] Table 29 N-2 and N- generation seed expansion information

Cultivation stage N-2 N-1 Batch No. 14 | 2 nd 3 rd Ist 2 nd 3 rd

Initial inoculation density (x 106 cells/mL) 0.32 0.22 0.2 0.44 0.54 0.52 Cell transfer density (x 106 cells/mL) 2.18 3.73 4.28 4.12 4.05 5.06 Cultivation time (day) 3 4 4 3 3 3 Cultivation volume (mL) 400 400 400 1790 1530 1775 Number of vessels 2 2 2 1 1 1 Cultivation vessel Shaking flask Reactor Vessel volume (L) 1 3

[260] Shaker parameter settings were shown in Table 25 (general parameter settings of a shaker).

[261] Sample collection plan

[262] Samples were collected from N-2 generation shaking flasks every 3 d to 4 d to detect viable cell density, cell viability, and cell diameter. Samples were collected from N-i generation reactors every day to detect viable cell density, cell viability, cell diameter, and biochemical parameters (pH, pO2, pCO2, Gln, Glu, Gluc, Lac, NH4 , Na+, K, Ca2 , and Osmo).

[263] Fed-batch cultivation

[264] Cultivation volume and inoculation density

[265] Table 30 Basic information of fed-batch cultivation

Cultivation batch No. 1s 15L 2 nd 15L 3 rd 15L Basic medium SFM4:CB5 (volume ratio: 9:1) Initial inoculation density (x 106 cells/mL) 0.81 0.87 0.93 Cultivation dilution ratio 5 4.7 5.4 Initial cultivation volume (mL) 7000 6940 6950 Reactor brand Broadly James Tank volume (L) 15

[266] Cultivation process settings

[267] Table 31 Reactor parameter control at the fed-batch cultivation stage

Cultivation batch No. 1s 15L, 2nd 15L, 3 rd 15L pH 6.9±0.2 DO (%) 40 Temperature 35 0 C Stirring speed (RPM) 160 Introduction of air at surface (L/min) 0.2 Introduction of air at the bottom (L/min) 0.07 Introduction of oxygen at the bottom (L/min) 0-1 Introduction of carbon dioxide at the bottom 0-0.2 (L/min) Ventilation mode 6-hole L-shaped ventilation (air hole diameter: S1mm) Other

[268] Feeding table

[269] Table 32 Feeding at the cultivation stage

Cultivation batch 1s 15L 15L 2 nd 3 rd 15L No. Fed-batch time (day) Fed-batch and fed-batch ratio 4 7% 2 x 104 (with 50 g/LFMO12), 0.28% FG01 7 10% 2 x 104 (with 50 g/LFM12), 0.4% FG01 9 10% 2 x 104 (with 50 g/LFMO12), 0.4% FGO1 11 10% 2 x 104 (with 50 g/LFM012), 0.4% FG01

[270] Sugar supplement strategy

[271] A glucose content was detected every day, and when the glucose content was lower than 3 g/L, a 400 g/L glucose stock solution was fed until the glucose content was 3 g/L to 5 g/L.

[272] Harvest standard

[273] Cells were harvested on day 15 of cultivation or harvested when a cell viability was lower than 50%.

[274] The sample collection and retention plan was shown in Table 26 (general sample collection and retention plan).

[275] Experimental results

[276] Cell growth

[277] It can be seen from the viable cell density data that the change trends over time for the three batches of 15 L experiments were basically the same. The highest viable cell densities all appeared on day 11, respectively of 27.06 x 106 cells/ml, 29.47 x 106 cells/ml, and 28.02 x 106 cells/ml, and the viable cell densities for the 3 batches of experiments on day 15 when the cells were harvested were basically the same, about 17 x 106 cells/ml. Viable cell density curves were shown in FIG. 12.

[278] It can be seen from the cell viability data that the cell viability change trends for the 3 batches of experiments were basically the same, and the cell viabilities at harvest were 66.8%, 59.8%, and 63.8%, respectively. Cell viability curves were shown in FIG. 13.

[279] Cell metabolism

[280] It can be seen from the glucose concentration data that the change trends for the 3 batches of 15 L experiments were basically the same; and when the glucose content was lower than 3.0 g/L after day 11, sugar was supplemented until a final glucose concentration was 3.0 g/L to 5.0 g/L. Glucose concentration curves were shown in FIG. 14.

[281] It can be seen from the lactate concentration data that the change trends for the 3 batches of L experiments were basically the same, and lactate concentrations for the 3 batches at harvest were 2.22 g/L, 2.45 g/L, and 2.12 g/L, respectively. Lactate concentration curves were shown in FIG. 15.

[282] Protein yield results

[283] It can be seen from the protein yield data that the protein yields for the 3 batches were basically the same, respectively of 2.35 g/L, 2.39 g/L, and 2.45 g/L. Protein yield curves were shown in FIG. 16.

[284] Protein quality results

[285] The protein quality results were all quoted from the test data obtained after the three-step purification of the downstream protein purification department. It can be seen from the CEX test data that the CEX acid peak areas for the 3 batches of 15 L experiments at harvest on day 15 were all lower than 13.3%. CEX test results were shown in Table 33.

[286] Table 33 Protein CEX test results

Test results Sample No. Main peak % Acidic peak % Basic peak

% JNY-INV 45.7 8.7 45.6 1' 15L 57.9 9.3 32.8 JNY-INV 45.6 9.1 45.3 2 nd 15L 57.6 10.7 31.7 JNY-INV 45.8 8.7 45.5 3 rd 15L 58.4 9.9 31.8

[287] It can be seen from glycoform distribution data that the GOF values for the 3 batches of 15 L experiments at harvest on day 15 were 47.4%, 48.2%, and 47.4%, respectively. Glycoform distribution test results were shown in Table 34.

[288] Table 34 Protein glycoform distribution test results

Test results GOF-GN (%) GO GOF Man GI GIF G2 G2F other( Sample No. /MAN3+F ( -5() () () )

JNY-I 0.8 1.0 37.4 3.0 2.7 40.3 NA 8.6 7.0 NV 1t 15L 0.2 0.1 47.4 0.4 0.5 40 NA 7.9 3.8 JNY-I 0.9 1.0 38.1 3.0 3.1 39.9 NA 8.2 6.7 NV

0.2 0.1 48.2 0.3 0.4 40.2 NA 8.0 2.8 15L JNY-I 0.9 1.1 37.2 4.0 2.9 38.4 NA 8.1 8.2 NV 3 rd 15L 0.2 0.2 47.4 0.5 0.6 39.4 NA 7.8 4.1

[289] Data in this experiment showed that, through the final cultivation process determined from the small-scale process, the cell growth and metabolism parameters for 3 batches of 15 L production were basically the same, where a final protein yield range was 2.35 g/L to 2.45 g/L, CEX acid peak areas were all lower than 13.3%, and GOF values in the glycoform distribution results were basically 47% to 48%, which verified the stability of the process. This process could be used for 200 L production.

[290] Through 3 batches of 15 L experiments, the stability of the process was verified. A determined reactor process was shown in the table below, and 3 batches of production would be conducted at a 200 L reactor scale subsequently. A reactor process finally determined was as follows:

[291] Table 35 Reactor parameter settings

Clone No. Reactor parameter settings pH Dissolved oxygen (%) Temperature (°C) JK-I 6.90±0.2 40 35

[292] Table 36 Reactor fed-batch experimental scheme

Item Content Basic medium SFM4:CB5 (volume ratio: 9:1) Feeding plan 2x104 (with 50 g/L FM012) FG01 Day 4 7% 0.28% Day 7 10% 0.4% Day 9 10% 0.4% Day 11 10% 0.4%

[293] Example 5 Purification process

[294] The temperature, pH, organic solvents, and the like in the separation and purification step all will affect the activity of a target protein. The sequential order of steps and process control will also affect the purity and activity of a target protein. Therefore, in a separation and purification process of a biosimilar drug, separation and purification conditions used should be as reasonable and mild as possible, and the operation steps should be minimized. In addition, biosimilar drugs are all derived from biologically active substances, so potential hazard factors carried on biosimilar drugs, especially viral factors, need to be strictly controlled.

[295] Through many experiments to explore a purification process, the present disclosure adopted a four-stage process for purification. A cell culture discharged from a tank was clarified by two stage deep filtration, sterilized by filtration, and then loaded on a Protein A affinity chromatography column; an eluate from the affinity chromatography was subjected to low-pH incubation for virus inactivation, then neutralized, and filtered; a pH of a resulting sample was adjusted, the sample was loaded on an anion exchange chromatography column, and an effluent from the anion exchange chromatography was collected; and a pH of the effluent was adjusted, then the effluent was loaded on a cation exchange chromatography column, and an eluate from the cation exchange chromatography was collected and subjected to concentration and liquid exchange. A sample obtained from the liquid exchange was subjected to final sterilization filtration to obtain a product stock solution.

[296] On the basis of the small-scale process, 3 batches of 200 L pilot scale production were conducted, key process parameters were screened and optimized to determine a pilot-scale purification process, and 3 batches of products were tested and compared with the original product. It is expected that the average purification yield shall be not lower than 65%, the purity of the stock solution shall not be less than 95%, the protein A, DNA, and HCP residues shall meet requirements, and the activity shall be qualified.

[297] Table 37 Abbreviations

Abbreviation Definition ChP Chinese Pharmacopoeia USP United States Pharmacopoeia GE General Electric Company EQ Equilibration CV Column Volume WFI Water for Injection DBC Dynamic Binding Capacity UF/DF Ultrafiltration/Diafiltration NWP Normalized Water Permeability TMP Transmembrane Pressure LVIN Low pH Virus Inactivation & Neutralization SEC Size Exclusion Chromatography cIEF Capillary Isoelectric Focusing HMW High Molecular Weight LMW Low Molecular Weight HCP Host Cell Protein AEX Anion Exchange Chromatography CEX Cation Exchange Chromatography CE Capillary Electrophoresis LMH Liters per Square Meter per Hour

[298] The chemical reagents used in the downstream process were shown in Table 5. Unless otherwise specified, all reagents shall meet the grades specified in Chinese Pharmacopoeia or United States Pharmacopoeia or meet equivalent grades.

[299] Table 38 Reagents used in the downstream process

Reagent Manufacturer Grade Sodium hydroxide Hunan Er-kang Pharmaceutical Co., Ltd. ChP Sodium chloride Jiangsu Qinfen Pharmaceutical Co., Ltd. ChP Tris base Suhua Pharmaceutical Co., Ltd. ChP

Glacial acetic acid Sichuan Jinshan Pharmaceutical Co., Ltd. ChP Citric acid Hunan Er-kang Pharmaceutical Co., Ltd. ChP Sodium acetate Taishan Xinning Pharmaceutical Co., Ltd. ChP % ethanol Hunan Er-kang Pharmaceutical Co., Ltd. ChP

[300] 1. Clarification of a cell culture

[301] A cell culture discharged from a tank in Example 1 or 2 was clarified by two-stage deep filtration, then subjected to sterilization filtration, and loaded on an affinity chromatography column.

[302] Materials and equipment:

[303] Pressure sensor: PENDO TECH Pressure MAT

[304] Peristaltic pump: LongerPump, BT300-2J

[305] Samples: Three batches of 10 L cell culture

[306] Equilibration buffer: 50 mM Tris-HAc, 150 mM NaCl, pH 7.4

[307] Depth filter: Millipore Pod DOHC, 2 x 540 cm2 (grade 1); Millipore Pod AlHC, 540 cm2 (grade 2)

[308] Sterilization filter: Sartopore 2 300, Sartorius

[309] Operation steps:

[310] The depth filters AlHC and DOHC were rinsed separately with WFI at 100 L/m2 and 600 LMH.

[311] The two DOHC were collected in parallel and then connected to the AlHC in series, and the depth filters were equilibrated with the equilibration buffer (at least 2 times a dead volume of the DOHC system) at 600 LMH until a pH of an AlHC effluent was determined to be consistent with that of the equilibration buffer.

[312] The filtration of a sample was started, and a feed rate was gradually increased. An initial filtrate of a volume 0.5 to 0.7 times a dead volume of the entire system at an AlHC effluent end was discarded, and then the effluent end was connected to a sterilization filter in series to collect a filtrate.

[313] The filtration continued, where a feed rate was kept at < 200 LMH, and a pressure of a depth filter at each level was monitored and kept at < 15 psi.

[314] After the filtration of the sample was completed, a feed solution remaining in a depth filter membrane was replaced with a buffer of a volume at least 2 times the dead volume of the entire system

[315] The depth filters were evacuated, and all filtrates were combined.

[316] The protein A-HPLC was used to determine the antibody content in a sample before and after the deep filtration, and then a yield was calculated.

[317] Results of the deep filtration experiment for the three batches of 10 L cell culture were shown in Table 39.

[318] Table 39

Number Oprain el ulur of e ellty( Protein SolidCellfiltration filtration Depth Deep Operation cellculture viability 106 content content* membrane fila

from tank cells/mL) (g/L) (%) capacity (A1HC L/m2

) Deep

thfiraton o PD1-UA1 66.8 2.35 126.7 of 10 L Deep filtration for PD2-UAl- 59.8 17.24 2.39 5.3 161.7 103.2 the third DAY15 batch of 10 L Deep filtration for PD3-UA8- 63.8 17.37 2.45 5.3 165.0 103.4 the fourth DAY15 batch of 10 L

[319] *The solid content refers to a solid content (%) in a sample obtained after two-stage centrifugation (1,000 g, 10 min + 8,000 g, 10 min).

[320] 2. Affinity chromatography

[321] A supernatant obtained from clarification and sterilization filtration was purified by the Protein A affinity chromatography packing MabSelect of GE, and an eluate was collected and sampled for detection.

[322] Materials and equipment

[323] Packing and chromatography column: MabSelect, 5.0 x 25 cm, CV 491 mL

[324] Equipment

[325] avant, 569951, GE

[326] pH meter and conductivity meter: Seven Excellence, Multi-parameter, 595606, Mettler Toledo

[327] Spectrophotometer: Nanodrop 2000, 520161, Thermo Scientific

[328] Samples: Three batches of cell culture supernatant subjected to clarification and sterilization filtration

[329] Buffer (shown in Table 40)

[330] Methods and results

[331] Details of an affinity chromatography process were shown in Table 40. Operation steps of this experiment:

[332] An AKTA system and pipelines were subjected to disinfection and depyrogenation

Itn treatments with 1 M NaOH (lasting for more than 30 min).

[333] The AKTA system and pipelines were rinsed to neutral with WFI, and each buffer pipeline was filled with a corresponding buffer.

[334] A sample was filtered and restored to room temperature to be ready for sample loading.

[335] The chromatography column was connected to the system and an operation process was prepared.

[336] The chromatography process was implemented, and an eluate was collected and sampled for detection. Nanodrop was used to determine a protein concentration, and a yield was calculated.

[337] At the end of the chromatography process, the chromatography column was removed. The system pipelines were rinsed with water and stored with 20% ethanol.

[338] Table 40 Affinity chromatography process

Step Buffer Flow rate CV Remarks (cm/h) Rinsing 50 mM Tris-HAc, 150 mM 250 2 NaCl, pH 7.4 Disinfection before 50 mM NaOH, 1 M NaCl 250 2 Contact time: 15 min use EQ 50 mM Tris-HAc, 150 mM 250 3 NaCl, pH 7.4 Loading 250 DBC 5 35 mg/mLpacking, retention time 5 min Rinsing 1 50 mM Tris-HAc, 150 mM 250 3 NaCl, pH 7.4 Rinsing 2 50 mM Na-Acetate, 1 250 3 MNaCl, pH 6.0 Rinsing 3 50 mM Na-Acetate, pH 6.0 250 2 Collection UV280: Elution 50 mM Na-Acetate, pH 3.5 250 150- 150 mAU/mm Regeneration 1 MHAc 250 2 Rinsing 50 mM Tris-HAc, 150 mM 250 2 NaCl, pH 7.4 Disinfection after 50 mM NaOH, 1 M NaCl 250 2 Contact time: 15 min use Rinsing 50 mM Tris-HAc, 150 mM 250 2 NaCl, pH 7.4 Storage 20% Ethanol 250 2

[339] Table 41 Summary of affinity chromatography

DB SEC CE C ollectio Main/ cIEF Main/ Residua HCP DNA Yiel Unit n volume HMW/ Acidic Reduce I Pro A (ppm (pg/mg d operation (g/L (CV) LMW /Basic(%) d (%) (ppm) (%) (%) Protein A 98.8/1.1/0. 56.4/16.5/27. 97.5 4.1 123 85.4 chromatograph 1 1 y for the first batch of 10 L Protein A 22.4 1.0 99.2/0.8/N 57.3/14.7/28. 97.9 < 1.6 272 151.94 92.3 chromatograph D 0 y for the third 21.4 1.0 99.2/0.7/N 57.5/12.8/29. 97.8 < 1.6 271 167.96 90.2 batch of 10 L D 8 Protein A 24.9 1.1 99.1/1.0/N 58.9/13.9/27. 97.7 < 1.6 178 175.88 92.4 chromatograph D 2 y for the fourth 20.7 1.0 98.9/1.1/N 57.7/14.7/27. 97.7 < 1.6 288 551.97 91.8 batch of 10 L D 5

[340] 3. Low-pH incubation for virus inactivation and neutralization

[341] An eluate obtained from the affinity chromatography was adjusted to a pH of 3.5 to 3.8, then incubated for 2 h to 4 h at room temperature for virus inactivation, and then neutralized to a pH of 5.5 to 5.8, and a neutralized product was subjected to sterilization filtration.

[342] Materials and equipment

[343] Equipment

[344] pH meter and conductivity meter: Seven Excellence, Multi-parameter, 595606, Mettler Toledo

[345] Spectrophotometer: Nanodrop 2000, 520161, Thermo Scientific

[346] Sample: eluates from affinity chromatography

[347] Titration solutions: 1 M Citric Acid (adjustment acid); 1 M Tris Base (adjustment alkali)

[348] Sterilization filter: Bottle Top Filter, Express Plus, 0.22 m, Millipore

[349] Methods and results

[350] 3 mL was taken from an eluate to determine a pH, and 1 M citric acid was added to adjust the pH to 3.5 to 3.8. An addition percentage was calculated based on an addition amount of the 1 M citric acid. According to the addition percentage, 1 M citric acid was added to the eluate in proportion under stirring to adjust pH. A resulting mixture was thoroughly mixed, and then 3 mL was taken from the mixture to measure a pH for confirmation. The sample was placed at room temperature, and timing was started. 2 h to 4 h later, 3 mL was taken and added with 1 M Tris Base to adjust a pH to 5.5 to 5.8. Then the 1 M Tris Base was added to the sample in proportion according to an addition percentage. A resulting mixture was thoroughly mixed, and then 3 mL was taken from the mixture to measure a pH for confirmation. A neutralized sample was subjected to sterilization filtration. Low-pH incubation for virus inactivation and neutralization for three batches of 10 L eluate were shown in Table 42.

[351] Table 42 Summary of low-pH incubation for virus inactivation and neutralization

Unit SEC Main/ cIEF Main/ CE Residual HCP DNA Yield a HMW/ Acidic Purity Pro A operation LMW /Basic(%) Reduced (ppm)

(%) (%) Virus inactivation and 98.6/1.4/ND 55.3/17.9/26.9 97.7 5.9 95 32.58 98.6 neutralization for the first batch of 10 L Virus inactivation and 98.9/1.0/ND 58.7/14.3/27.0 97.7 2.6 162 1.63 99.2 neutralization for the third batch of 10 L Virus inactivation and 99.1/0.9/ND 57.3/16.0/26.7 97.6 1.8 151 1.10 98.8 neutralization for the fourth batch of 10 L

[352] 4. Anion exchange chromatography

[353] A sample subjected to low-pH virus inactivation and neutralization was adjusted to pH 6.9 and then loaded for anion exchange chromatography. An effluent from the anion exchange chromatography was collected and sampled for detection.

[354] Materials and equipment

[355] Packing and chromatography column: Super Q 650M (TOSOH), 2.6 x 22.8 cm, CV 121 mL

[356] Equipment

[357] avant, 569951, GE

[358] pH meter and conductivity meter: Seven Excellence, Multi-parameter, 595606, Mettler Toledo

[359] Spectrophotometer: Nanodrop 2000, 520161, Thermo Scientific

[360] Samples: samples subjected to virus inactivation and neutralization, which were adjusted to pH 6.9 before loading

[361] Sterilization filter: Bottle Top Filter, Express Plus, 0.22 m, Millipore

[362] Buffer (shown in Table 43)

[363] Methods and results

[364] Details of an anion exchange chromatography process were shown in Table 43. Operation steps of this experiment (experimental results were shown in Table 44):

[365] An AKTA system and pipelines were subjected to disinfection and depyrogenation treatments with 1 M NaOH (lasting for more than 30 min).

[366] The AKTA system and pipelines were rinsed to neutral with WFI, and each buffer pipeline was filled with a corresponding buffer.

[367] A sample was filtered and restored to room temperature to be ready for sample loading.

[368] The chromatography column was connected to the system and an operation process was prepared.

[369] The chromatography process was implemented, and a protein effluent was collected and sampled for detection. Nanodrop was used to determine a protein concentration, and a yield was calculated.

[370] At the end of the chromatography process, the chromatography column was removed. The system pipelines were rinsed with water and stored with 20% ethanol.

[371] Table 43 Anion exchange chromatography process

Step Buffer Flow rate CV Remarks _____________(cm/h)

Disinfection 1 M NaOH 250 2 Retention: 60 min before use Pre-equilibrium 500 mM Tris-HAc, pH 7.2 250 2 Equilibrium 100 mM Tris-HAc, pH 6.9 250 3 DBC 5 189 mg/mL packing, retention time 5 Loading 250 min, start collection UV280: 50 mAU/mm End collection Rinsing 1 100 mM Tris-HAc, pH 6.9 250 2 UV280: 50 mAU/mm

Regeneration 50 mM Na-Acetate, 250 2 1 M NaCl, pH 6.0 Disinfection 1 M NaOH 250 2 Retention: 60 min after use Storage 0.1 M NaOH 250 2

[372] Table 44 Summary of anion exchange chromatography

SEC Main/ cIEF Main/ CE Residual Unit DBC HMW/ Acidic Purity Pro A HCP DNA Yield operation (g/L) LMW /Basic(%) Reduced (ppm) (ppm) (pg/mg) (%) (%) (%) AEX for

bathe fir 73.0 98.6/1.3/0.1 57.3/16.0/26.7 97.8 2.4 31 <0.30 99.5 L AEX for 155.8 98.8/1.2/ND 58.3/14.6/27.1 97.7 < 1.6 33 <0.29 99.4 the third batch of 10 L AEX for

98.9/1.1/ND 59.4/13.7/26.9 97.8 < 1.6 47 <0.29 99.0 bate of10 162.3 L

[373] 5. Cation exchange chromatography

[374] An effluent from the anion exchange chromatography was adjusted to pH 5.0 and loaded for cation exchange chromatography. An eluate from the cation exchange chromatography was collected and sampled for detection.

[375] Materials and equipment

[376] Packing and chromatography column: Poros XS, 2.6 x 25.4 cm, CV 135 mL (the first batch of 10 L); Poros XS, 5.0 x 21.8 cm, CV 428 mL (the third batch of 10L and the fourth batch of 10 L)

[377] Equipment

[378] avant, 569951, GE

[379] pH meter and conductivity meter: Seven Excellence, Multi-parameter, 595606, Mettler Toledo

[380] Spectrophotometer: Nanodrop 2000, 520161, Thermo Scientific

[381] Samples: effluents from the anion exchange chromatography, which were adjusted to pH 5.0 (when the third batch of 10 L was loaded, the sample flowed through as a pH was not adjusted, and the remaining sample and the flow-through sample were adjusted to pH 5.0 and then loaded)

[382] Sterilization filter: Bottle Top Filter, Express Plus, 0.22 tm, Millipore

[383] Buffer (shown in Table 45)

[384] 8.2 Methods and results

[385] Details of a cation exchange chromatography process were shown in Table 45. Operation steps of this experiment (experimental results were shown in Table 46):

[386] An AKTA system and pipelines were subjected to disinfection and depyrogenation treatments with 1 M NaOH (lasting for more than 30 min).

[387] The AKTA system and pipelines were rinsed to neutral with WFI, and each buffer pipeline was filled with a corresponding buffer.

[388] A sample was filtered and restored to room temperature to be ready for sample loading.

[389] The chromatography column was connected to the system and an operation process was prepared.

[390] The chromatography process was implemented, and an eluate was collected and sampled for detection. Nanodrop was used to determine a protein concentration, and a yield was calculated.

[391] At the end of the chromatography process, the chromatography column was removed. The system pipelines were rinsed with water and stored with 20% ethanol.

[392] Table 45 Cation exchange chromatography process

Step Buffer Flow rate CV Remarks _______________(cm/h)

Disinfection 1 MNaOH 250 2 Retention: 60 min before use EQ 50 mM Na-Acetate, pH 250 3 5.0 Loading 250 DBC <65 mg/mLpacking, retention time 5 mm Rinsing 1 50 mM Na-Acetate, pH 250 2 5.0 Rinsing 2 50 mM Na-Acetate, 65 250 5 mM NaCl, pH 5.0 Rinsing 3 50 mM Na-Acetate, pH 250 2 5.0 Elution 50 mM Na-Acetate, 140 250 Collection UV280: 150 -150 mM NaCl, pH 5.0 mAU/mm Regeneration 50 mM Na-Acetate, 1 250 2 M NaCl, pH 6.0 Disinfection after 1 M NaOH 250 2 Retention: 60 min use Storage 0.1 M NaOH 250 2

[393] Table 46 Summary of cation exchange chromatography

Collect SEC EF M cain/ E Residual HCP DNA Yield Unit DBC vlume HM/ Acidic Puiy Pro A C DN Yil operation (g/L) (CV) LMW /Basic(%) Reduced (ppm) (pg/mg) (%) (%) (%) CEX for thefirst 55.8 1.4 99.2/0.8/ND 58.8/14.4/26.9 98.2 < 1.6 <1 / 85.6 batch of 10 L CEX for thethird 43.2 1.4 99.5/0.5/ND 58.6/15.0/26.5 98.0 < 1.6 1 < 0.20 83.0 batch of 10 L CEX for the fourth 44.4 1.5 99.0/1.0/ND 59.7/13.8/26.5 98.2 < 1.6 1 < 0.19 87.3 batch of 10 L

[394] Table 47 Glycan-HPLC results and CEX-HPLC results of cation exchange chromatography products

Glytan-HPLC result CEX-HPLC result Unit operati Sample GO GO GO Ma G1 G1 G2 G2 Othe Main Acidic Basic ID F- (% F n -5 (% F (% F rs peak peak peak on GN/) (% (% ) (% )(%(%) % % %

MA ) )

) N 3+1 F (%) CEX INV(10341 0.9 1.0 38. 3.0 3.1 39. N 8.2 6.7 45.7 8.7 45.6 for the 49) 1 9 A first4740 N batch of SD723G04 0.2 0.1 04 .4 0.5 40. N 7.9 3.8 57.9 9.3 32.8 10L CEX INV(10341 0.9 1.0 38. 3.0 3.1 39. N 8.2 6.7 45.6 9.1 45.3 for the 49) 1 9 A third 48. 40. N batchof SD823D01 0.2 0.1 2 0.3 0.4 2 N 8.0 2.8 57.6 10.7 31.7 10L CEX INV(10341 0.9 1.1 37. 4.0 2.9 38. N 8.1 8.2 45.8 8.7 45.5 for the 49) 2 4 A fourth 47 39 N batch of SD829D01 0.2 0.2 4 0.5 0.6 4 A 7.8 4.1 58.4 9.9 31.8 10L

[395] *The sample is a filtrate obtained by subjecting an eluate from the cation exchange chromatography to dilution and then to nanofiltration.

[396] 6. Concentration and dialysis (UF/DF)

[397] An eluate from the cation exchange chromatography was subjected to concentration and buffer exchange. After the concentration and buffer exchange were completed, a protein concentration in the stock solution was adjusted to 19 mg/mL to 21 mg/mL, and then the stock solution was subjected to final sterilization filtration.

[398] Materials and equipment

[399] Equipment

[400] Peristaltic pump: Longer Pump, BT300-2J/YZ1515x, BT300-2J/YZ2515x

[401] Pressure monitor: Pendo TECH, Pressure MAT

[402] pH meter and conductivity meter: Seven Excellence, Multi-parameter, 595606, Mettler Toledo

[403] Spectrophotometer: Nanodrop 2000, 520161, Thermo Scientific

[404] Samples: eluates from the cation exchange chromatography

[405] Buffer

[406] UF/DF equilibration and dialysis buffer: 6.4 g sodium acetate trihydrate/kg solution, 5.8 g sodium chloride/kg solution, 0.182 g glacial acetic acid/kg solution, pH 5.8, osmotic pressure: 276 mOsm/kg.

[407] Membrane disinfectant: 1 M NaOH

[408] Membrane preservation solution: 0.1 M NaOH

[409] Sterilization filter: Bottle Top Filter, 0.22 [m, PES, Corning

[410] Ultrafiltration membrane and fixture: Pellicon XL Biomax 30, 50 cm2 , CAT No. PXB030A50, Lot No. C2MA64012-030 (the first batch of 10 L was subjected to concentration and liquid exchange); Pellicon 2mini 30k, 0.1 m2 , CAT No. P2B030A01, Lot No. C2PA14764-001, fixture: Millipore ZJMP-14-001 (the third batch of 10 L and the fourth batch of 10 L were subjected to concentration and liquid exchange)

[411] Methods and results

[412] Details of a concentration and liquid exchange method were shown in Table 48. UF/DF operation steps of this experiment (experimental results were shown in Tables 49 and 50)

[413] A membrane was rinsed with WFI (new membranes were rinsed at more than 80 L/m2 , and used membranes were rinsed at more than 20 L/m2 ).

[414] 1 M NaOH was used for disinfection before use (circulating for 1 h).

[415] The membrane was rinsed with WFI to neutral and NWP determination was conducted. At a feed flux of 360 LMH, a feed end pressure of 1.0 bar, and a backflow end pressure of 0.5 bar, a water flow rate at the permeate end was determined to calculate the NWP.

[416] Water in pipelines and membranes was evacuated to determine a dead volume of the system.

[417] The membrane was equilibrated with an equilibration buffer of a volume at least 2 times the dead volume of the system. The pH and electrical conductivity were determined for effluents at the backflow end and permeate end, which needed to be consistent with that of the equilibration buffer.

[418] A sample was subjected to concentration and liquid exchange, and during this process, a feed flux was no more than 360 LMH, and a pressure at the backflow end was adjusted to keep a pressure at the feed end at no more than 20 psi and keep a transmembrane pressure at no more than psi.

[419] After the liquid exchange was conducted by at least 6 times, appropriate over-concentration was required if necessary, then a valve of a permeate port was closed, and circulation was conducted for more than 5 min with the dialysis buffer of a volume at least 2 times the dead volume of the system. The membrane was evacuated, and all products were recovered in a product storage tank.

[420] The membrane was rinsed with WFI.

[421] 1 M NaOH was used for disinfection after use (circulating for 1 h).

[422] The membrane was rinsed with water and stored with 0.1 M NaOH.

[423] A product obtained from the concentration and liquid exchange was adjusted to a concentration of 19 mg/mL to 21 mg/mL with an appropriate amount of dialysis buffer, and then filtered with a sterilization filter, and a UF/DF yield was calculated.

[424] Table 48 UF/DF process Step Description

Membrane rinsing Rinse with WFI Disinfection before use Circulate for 1 h with1 M NaOH Membrane rinsing and NWP determination NWP determination before use Equilibration Rinse the membrane with an equilibration buffer

Lioceni n IMP TF) 5 15 psi, pressure at the feed end 5 20 psi Recycling of product Recycle a product with an equilibration buffer Membrane rinsing Rinse with WFI Disinfection after use Circulate for 1 h with1 M NaOH Membrane rinsing and NWP determination NWP determination after use Storage Store the membrane with 0.1 M NaOH

[425] Table 49 Stock solution test result 1 (UF/DF summary)

SEC CEX UF/D Unit Stock F Protein Main/ HPLC Residu HC DNA Endoto operate soluti concentrat HMW Main/ al Pro P (pg/ xin on capaci ion / Acidic A (pp (EU/mg d on No. ( mg/mL) LMW /Basic( (ppm) m)

) / %(%)) UF/DF for the 97.6/2 first SD09 241.7 19.91 4 ND TBD TBD TBD TBD TBD 96.8 batch of 1OL UF/DF for the 98.9/1 third SD12 152.3 19.98 2/ ND TBD TBD TBD TBD TBD 97.6 batch of 1OL UF/DF for the 99.5/0 fourth SD11 164.1 20.60 6/ND TBD TBD TBD TBD TBD 99.9 batch of 1OL

[426] Table 50 Stock solution test result 2 (UF/DF summary)

CE Cell RP- CHIEF Main/ C PuCE SDS- SDSy SDS- PAGE Cl Unit Stock P Acidic /Basic Purity Purity PAGE PAGE Based operation solution No• mapping AcReduced reduced Reduced reduced Bioassay redce (%)rdue

UF/DF

TBD 59.6/13.5/26.9 TBD TBD TBD TBD TBD forb tch SD09 of 10 L UF/DF for the SD12 TBD 59.8/13.5/26.6 TBD TBD TBD TBD TBD third batch of 1OL UF/DF for the fourth SD11 TBD 58.1/12.2/29.7 TBD TBD TBD TBD TBD batch of 1OL

[427] It can be understood that the above implementations are merely exemplary implementations used to illustrate the principle of the present disclosure, and the present disclosure is not limited thereto. Various modifications and improvements can be made by those of ordinary skill in the art without departing from the spirit and essence of the present disclosure, and these modifications and improvements are also considered as falling within the protection scope of the present disclosure.

A r%

Claims (6)

WHAT IS CLAIMED IS:

1. An establishment method of a cell line for preparing an anti-epidermal growth factor receptor (EGFR) fully-humanized monoclonal antibody (mAb), wherein dihydrofolate reductase (DHFR) deficient Chinese hamster ovary (CHO) cell lines CHO/DHFR- are subjected to POOL screening after undergoing transfection, and the POOL screening comprises: stage 1, using an HT-free SFM4CHO medium to screen out a cell line integrated with a heavy chain (HC) gene, and using Blasticidin in the medium to screen out a cell line integrated with a light chain (LC) gene; and stage 2, adding MTX to increase copy numbers of the LC gene and the HC gene that are integrated into a genome of a cell line, thereby increasing an expression level.

2. The establishment method of a cell line for preparing an anti-EGFR fully-humanized mAb according to claim 1, wherein at stage 1 in the POOL screening, the medium has a GlutaMAX-I concentration of 4 mM and a Blasticidin concentration of 10 mg/l, and cells are passaged twice a week until a cell viability is restored to more than 85%; and at stage 2, the medium has a GlutaMAX-I concentration of 4 mM, a Blasticidin concentration of 10 mg/, and an MTX concentration of 500 nM, and cells are passaged twice a week until a cell viability is restored to more than 90%.

3. The establishment method of a cell line for preparing an anti-EGFR fully-humanized mAb according to claim 2, wherein after the POOL screening is completed, clonal expansion is conducted by 2X103 fed-batch culture; and during the 2X103 fed-batch culture, FMO12 has a concentration of 50 g/l and sugar is supplemented to a final concentration of 10 g/l to 12 g/l in each feeding.

4. The establishment method of a cell line for preparing an anti-EGFR fully-humanized mAb according to claim 3, wherein after the fed-batch culture, plated culture is conducted as follows: adding cells in a centrifuge tube with a semi-solid medium at a calculated density of 300 cells/ml, thoroughly mixing and adding a resulting cell suspension to a culture plate at an amount of 2.5 ml/well, and statically cultivating the cells in an incubator with 36.5°C and 6% CO 2 .

5. The establishment method of a cell line for preparing an anti-EGFR fully-humanized mAb according to claim 4, wherein a preparation method of the semi-solid medium used for the plated culture comprises: 24 h before the plated culture, transferring 90 ml of Clone Medium CHO DHFR from -40°C to 2°C to 8°C in a refrigerator; adding 2 ml of GlutaMAX-I, 1 ml of Clone Detect antihuman FITC, and 2 ml of Clone XL Reagent to the ready Clone Medium CHO DHFR, and adding MTX and Blasticidin at an amount such that the MTX and Blasticidin have concentrations respectively of 500 nM and 10 mg/i in 100 ml of the semi-solid medium; and adjusting a volume of a resulting mixture to 100 ml with a host cell supernatant.

6. The establishment method of a cell line for preparing an anti-EGFR fully-humanized mAb according to claim 5, wherein during an establishment process, cells are cultivated under the following conditions: temperature: 36.5°C, humidity: 75% to 85%, carbon dioxide concentration: 6%, rotation radius: 2.5 cm, rotational speed: 110 rpm to 225 rpm, and volume: 20 ml to 30 ml.